m6A‐mediated alternative splicing coupled with nonsense‐mediated mRNA decay regulates SAM synthetase homeostasis

Alternative splicing of pre‐mRNAs can regulate gene expression levels by coupling with nonsense‐mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS‐NMD) in an organism, we performed long‐read RNA sequencing of poly(A) + RNAs from an NMD‐deficient mutant strain of Caenorhabditis elegans , and obtained full‐length sequences for mRNA isoforms from 259 high‐confidence AS‐NMD genes. Among them are the S‐adenosyl‐L‐methionine (SAM) synthetase ( sams ) genes sams‐3 and sams‐4 . SAM synthetase activity autoregulates sams gene expression through AS‐NMD in a negative feedback loop. We furthermore find that METT‐10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo , and specifically methylates the invariant AG dinucleotide at the distal 3′ splice site (3′SS) in␣vitro . Direct RNA sequencing coupled with machine learning confirms m 6 A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m 6 A modification at the 3′SS of the sams genes. SYNOPSIS Alternative splicing (AS) of pre‐mRNAs can regulate gene expression levels by coupling with nonsense‐mediated mRNA decay (NMD). Here, assessment of the organismal substrate repertoire of this process reveals SAM synthetase ( sams ) gene expression regulation via m 6 A modification in C. elegans . Long‐read sequencing of poly(A)+ RNAs from an NMD‐deficient mutant strain reveals 259 high‐confidence AS‐NMD genes, including the SAM synthetase ( sams ) genes. SAMS activity negatively autoregulates AS‐NMD of the sams genes in␣vivo . Direct RNA sequencing coupled with machine learning reveals m 6 A modification of endogenous sams mRNAs at the distal 3′ splice sites. Methyltransferase METT‐10 catalyzes m 6 A modification specifically at the distal 3′ splice site of sams pre‐mRNA in␣vitro . METT‐10 regulates alternative splicing of the sams genes in␣vivo . Graphical Abstract Full‐length sequences of natural NMD isoforms in C. elegans are elucidated by direct RNA sequencing. SAM synthetase homeostasis is regulated via pre‐mRNA splicing of the sams genes through m 6 A modification at the invariant AG of the distal 3′SS.